Various types of immunoassay involve attaching fluorescent labels either directly or indirectly to a species of interest, for example a microorganism. Animal, plant and microbial cells contain significant amounts of aromatic compounds, many of which are intrinsically fluorescent (autofluorescent) when excited at an appropriate wavelength. The range of fluorescent substances of biological origin described in the biochemical literature is large and includes chlorophyll, haemoglobin and protein. When these autofluorescent materials have high quantum yields (typically >0.02) they can present a problem of spurious fluorescence against which fluorescent labels of interest must be detected.
Autofluorescence can occur throughout the visible spectrum and is typically a short-lived phenomena with a lifetime (τ) measured in nanoseconds. Autofluorophores may have a fluorescence lifetime (τ) ranging from 1 to 100 nanoseconds and synthetic fluorophores are available with τ more than 20,000 times longer (millisecond lifetimes).
A number of methods have been applied to reduce the severity of the problem of autofluorescence and time-resolved fluorescence microscopy (TRFM) is a proven technique that can largely eliminate its effects. Time-resolved fluorescence microscopes can roughly be divided into two types: those designed to discriminate fluorophores with very short lifetimes (nanoseconds) operating in the frequency domain, and instruments that exploit the time domain and employ fluorophores with longer fluorescence lifetimes (microseconds and greater). Time-resolved fluorescence techniques have been developed to exploit the (comparatively) long fluorescence lifetimes (>300 μs) observed with lanthanide chelates. Europium and terbium chelates are often employed in fluorescent labels due to their useful visible emission (red and green respectively), however the antenna molecule used to transfer energy to the chelated lanthanide ion typically requires excitation in the UV range (320 to 360 nm). Time-resolved fluorescence methods employ pulsed-excitation of the fluorophore, followed by a gate-delay phase to permit decay of short-lived fluorescence. A disadvantage with the use of flashlamps as the excitation source is that the duration of the gate-delay period must be extended to ensure that light from the decaying arc plasma has decayed to zero. Flashlamp plasma can persist for hundreds of microseconds even for lamps with rated arc duration of 1 or 2 μs and the faint emission from the plasma can obscure the weak emission from most fluorophores for a significant time.
Detection of rare organisms that may occur in the early stages of infection or in cases where the organisms are generally unculturable is very difficult when even moderate autofluorescence is present. Suppression of background autofluorescence results in much greater detection success and consequently fluorescence microscopy techniques may be applied in circumstances where they are currently not used due to the presence of autofluorophores.
No current techniques eliminate the problem of autofluorescence under all circumstances, however the use of narrow passband filters is the most common and readily available technique. In this manner a specific narrow excitation spectrum is used to excite fluorescence from the probe with minimal competing fluorescence from autofluorophores. The emission spectrum is also selectively captured within a narrow passband to limit the impact of autofluorescence. The method relies on the availability of suitable fluorophores with spectral characteristics sufficiently different from the principal interfering autofluorophore.
Conventional methods of generating a pulsed excitation source for time-resolved fluorescence detection have often relied on the use of mechanical shutters (chopper wheel). Although systems employing this techniques have the advantage of suppressing autofluorescence in real-time, they are not ideal. They have disadvantages of low pulse repetition speed, slow rise and fall-time of the excitation pulse, sensitivity to vibration due to the very rapid rotation of the chopper wheel and uneven illumination across the viewing plane (sunrise-sunset effect). Mechanical systems are also generally found to be less reliable than electronic systems. A second method of blocking light from the excitation source is based on ferro-electric liquid crystals that act to shift the plane of polarization of light reaching the observer. The liquid crystal is sandwiched between two polarizing elements that are oriented to block the passage of any light unless it is rotated into the plane of the analyser. The optical shutter is controlled by applying a voltage to the ferro-electric liquid crystal so that the plane of polarization is rotated in the correct orientation to pass through the exit analyser. A disadvantage of ferro-electric liquid crystal shutters is the relatively slow operation of the shutter, with a period of 80 microseconds required before the shutter has opened or closed to 90% of maximum.
UV excitation energy should ideally be delivered with very rapid (sub-microsecond) rise and fall times for maximum efficiency of an instrument. Solid-state sources are easily switched at low voltages with nanosecond accuracy, however until recently no devices were available that operate in the required region of the near UV spectrum, for example in the range of 330-370 nm. Prior to the appearance of solid-state UV sources on the market, a number of sub-optimal methods of generating pulsed UV were employed. These included mechanical choppers interrupting a UV rich light source, acousto-optical switches to deflect laser UV sources and rapid discharge flashlamps (as in our prototype instrument).
Detection of the faint fluorescence from the time-resolved fluorescence probe typically required a cooled CCD camera integrating a faint signal over a period of minutes, or the use of an image intensified, time-gated CCD camera. The latter instrument has the advantage of speed, however resolution is inferior to the cooled CCD camera and the cost is a significant factor in the final expense of a TRFM system.
There is therefore a need for a fluorescence detection system with fast switching speeds, preferably in the nanosecond timescale. There is a further need for a fluorescence detection system with improved sensitivity, so that infrequent fluorescence events may be detected. The systems would preferably be relatively inexpensive.